THE APOBEC IN APOBEC MUTAGENESIS ABSTRACT Mutations drive the initiation and progression of cancer. The leading druggable source of mutation in cancer, cytosine deamination by a subset of the nine-membered APOBEC family of DNA deaminase enzymes, leaves a distinct mutation signature on the cancer genome. This signature is characterized as C-to-T and C-to- G mutations in a TCA/T trinucleotide context, and thus APOBEC-dependent mutations can be resolved computationally from other processes of mutation in clinical next-generation tumor sequencing datasets. While specific APOBEC3 (A3) enzymes have been implicated as the main progenitors of this mutation signature (namely, A3B, A3H, and A3A), the literature is full of conflicting data and it is not clear which of these enzymes contributes the most and whether other A3 enzymes may also contribute. I propose to resolve this debate through two specific aims. First, I will develop an A3-null haploid cell model system in order to systematically assay the genomic impact of individual APOBEC family members and document enzyme-specific mutation signatures. Second, I will use CRISPR to create individual A3 knockout clones in APOBEC signature-positive cell lines to understand how individual A3 enzymes contribute to functional resistance mutations and clonal evolution. In addition, I will use both of these wet lab experimental data sets to inform computational pipelines to reconstruct the APOBEC signature in publicly available tumor sequencing datasets. Collectively, I anticipate that these experiments will determine the APOBEC enzyme (or enzymes) responsible for mutagenizing cancer genomes across numerous cancer types. In turn, this fundamental knowledge is expected to inform the development of future diagnostics and therapies to prevent this mutational process and poor clinical outcomes including drug resistance and metastasis.